Method for deriving epitopes

ABSTRACT

A method of generating a nucleic acid species which are immunologically cross-reactive with non-nucleic acid immunogens is disclosed. The method comprises combining an antigen binding protein which binds said immunogen with a degenerate pool of nucleic acid species, and then recovering a nucleic acid species bound by said antigen binding protein from said degenerate pool. Also disclosed are the nucleic acid species so made, along with the use thereof for tagging molecules for immunological detection, for detecting antibodies to predetermined non-nucleic acid immunogens, for blocking complex formation between an antigen binding protein and a non-nucleic acid immunogen, and for inducing an immune response to the immunogen in a human or animal subject. Preferred immunogens are peptides and preferred antigen binding proteins are antibodies.

RELATED APPLICATIONS

[0001] This Application is a Continuation-in-Part of copendingapplication of Jack D. Keene, Daniel J. Kenan, and Donald E. Tsai filedSep. 11, 1992 (attorney docket number 5405-69), the disclosure of whichis incorporated by reference herein in its entirety.

[0002] This invention was made with government support under a grantfrom the National Institutes of Health. The government has certainrights to this invention.

FIELD OF THE INVENTION

[0003] This invention relates to methods of making epitopes and nucleicacids embodying the epitopes so made.

BACKGROUND OF THE INVENTION

[0004] It is desireable to be able to probe and dissect the precisesites of antigen-antibody interaction. It is also desireable to findnovel ways to detect antibodies and to inhibit specific antibody-antigeninteractions. Furthermore, methods are needed that allow one to purify amonospecific antibody from a polyclonal serum without having to firstpurify the antigen.

[0005] It has not heretofore been possible to produce distinct andgenerally useful epitopes which react with a given antibody except inthe case of epitope libraries. These peptide libraries depend uponexpression of a random set of epitopes within the context of a largerprotein. See, e.g., Scott et al., Science 249, 386 (1990); Cwirla etal., Proc. Natl. Acad. Sci. USA 87, 6378 (1990). This approach isrestricted because it offers only proteinaceous ligands and ispotentially compromised in contexts other than the fusion protein. Inaddition, “combinatorial peptide libraries” have been described whichapply sequential positional determinations. See Houghten et al., Nature454, 84 (1991). This procedure requires evaluation of the selectedligand at each step which, in turn, requires an exponential effort todefine and select a specific epitope. Further, methods for screeningdegenerate pools of peptide sequences have been used which are notlimited by proteinaceous context but are limited for logistical reasons(e.g., sophisticated synthesis and detection instruments are required).See Fodor et al., Science 251, 767 (1991); Geysen et al., Proc. Natl.Acad. Sci. USA 81, 3998 (1984).

[0006] S. Deutscher and J. Keene, Proc. Natl. Acad. Sci. USA 85, 3299(1988) describe the selection and amplification of a nucleic acid ligandon U1 RNA from a randomized pool of nucleic acids (see also J. Wiluszand J. Keene, J. Biol. Chem. 261, 5467 (1986)). L. Gold and C. Tuerk,Nucleic Acid Ligands, PCT Appln. Publn No. WO 91/19813 (26 Dec. 1991),describe the “evolution” of nucleic acid ligands and nucleic acidcompounds refered to as “nucleic acid antibodies” (see also C. Tuerk andL. Gold, Science 249, 505-510 (1990)). A. Ellington and J. Szostak,Nature 346, 818-822 (1990), describe the binding of RNA molecules toorganic dyes. D. Tsai et al., Nucleic Acids Research 19, 4931-4936(1991), describe the binding of the U1-snRNP-A protein to specific RNAsequences in a degenerate pool of transcripts. D. Bartel et al., Cell67, 529-563 (1991), describe the binding of the Rev protein of HIV-1 toa nucleic acid pool.

[0007] There has not heretofore been described a method by which anantibody can be employed to derive a nonproteinaceous mimetic ligandthat binds to the same site on the antibody to which the originalantigen bound.

SUMMARY OF THE INVENTION

[0008] A first aspect of the present invention is a method of generatinga nucleic acid molecule which is immunologically cross-reactive with animmunogen, which immunogen is not a nucleic acid (e.g., a peptide). Themethod comprises combining an antigen binding protein which binds theimmunogen (e.g., an antibody, a T cell receptor) with a degenerate poolof nucleic acid species, and then recovering a nucleic acid speciesbound by said binding protein from the degenerate pool.

[0009] A second aspect of the present invention is an isolated nucleicacid which inhibits complex formation between an antigen binding proteinand an immunogen, which immunogen is not a nucleic acid. In oneembodiment, the nucleic acid inhibits complex formation between a selfpeptide autoantigen and an antigen binding protein.

[0010] The foregoing and other objects and aspects of the presentinvention are explained in detail in the drawings herein and thespecification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a schematic diagram of a process of the instantinvention.

[0012]FIG. 2 shows D10 DNA construct (upper)(SEQ ID NO:1) and thedegenerate RNA transcript (lower) (SEQ ID NO:2) used to select the D10RNA epitope (SEQ ID NO:3). Degenerate nucleotide positions in thepredicted RNA secondary structure are represented by “N.” BamHIrestriction sites, the T7 promoter, complementary regions representingU1 stem II and the D10 loop sequence are indicated.

[0013]FIG. 3 shows the g10 fusion peptide used as an epitope tag. PanelA: Western blot of E. coli extracts containing various U1-A proteinconstructs. All extracts were produced from induced E. coli cellscontaining recombinant pET-8c T7 expression vectors in accordance withknown techniques. See F. Studier et al., Meth. Enzym. 185, 60-89 (1991).U1-A cDNA was cloned into pET-8c either attached or not attached to theg10 peptide. Lanes: 1, 5 and 9: the pET-8c vector alone; 2, 6 and 10:g10-U1-A protein; 3, 7 and 11: U1-A protein; 4, 8 and 12: total HeLacell extracts. Lanes 1-4 were probed with pre-immune rabbit serum; lanes5-8 were probed with anti-g10 serum; and lanes 9-12 were probed with aU1/U2-specific autoimmune serum (patient EW). EW reacts with U1 RNA,U1-70K (70 kDa), U1-A (31 kDa) and U2-A′ (28.4 kDa) (lane 12). Panel B:Immunoprecipitation and competition analysis of [³⁵S]-methionine-labeledU1-A and g10-U1-A proteins expressed by in vitro transcription, andsubsequent translation in rabbit reticulocyte lysates. Lanes: 1 and 2show the amount of U1-A and g10-U1-A translation product, respectively,added to each immunoprecipitation reaction; 3: blank; 4 and 5: U1-A andg10-U1-A precipitated with serum EW (reactive with U1-A); 6 and 7: U1-Aand g10-U1-A, respectively, precipitated with anti-g10 serum. In lanes8-12, g10-U1-A was precipitated with anti-g10 serum in the presence ofvarying amounts of competitors as follows: lanes 8-11 contained the g10peptide at concentrations of 27.8, 83.3, 250 and 750 nM, respectively;lane 12 contained a control peptide (sequence GKSRGFAFVEFK-amide) (SEQID NO:4) at a concentration of 25 mM. The faint lower band in all lanesrepresents either a degradation product or a premature translationtermination product and is seen consistently in U1-A translations.

[0014]FIG. 4 shows the D10 RNA epitope binds specifically to g10antibodies. Various antisera were used to precipitate either [³²P]labeled in vitro transcribed D10 RNA or control transcripts. Bound RNAwas analyzed on a denaturing 6% polyacrylamide gel by autoradiography.Lanes 1-4 are precipitations of D10 RNA with the following antisera: 1,anti-g10 serum; 2, preimmune serum; 3, no antibody; 4, anti-g10 serum.Other transcripts were precipitated with the anti-g10 serum as follows:5, a control RNA identical to the D10 epitope but with loop sequence5′-CACCAUAUAA-3′ (SEQ ID NO:5); 6, an unrelated RNA; 7, an RNAcontaining the loop sequence 5′-CUGACCCCGU-3′(SEQ ID NO:6); 8-11,supernatants from the immunoprecipitations shown in lanes 4-7, dilutedto approximate radioactive equivalents.

[0015]FIG. 5 shows competition analysis of g10 peptide and D10 RNAepitopes for binding by the g10 antibodies. [³²P] labeled in vitrotranscribed D10 RNA or [³⁵S] labeled in vitro translated g10 fusionprotein (g10-U1A) was immunoprecipitated by the anti-g10 serum in thepresence of various competitors. D10 RNA and g10 fusion protein bound inthe immunoprecipitations were analyzed using denaturing polyacrylamidegels and autoradiography. Panel A: D10 RNA immunoprecipitations with: 1,no competitor; 2, no g10 antiserum added; 3, 125 mg of g10 peptide; 4,125 mg of bovine serum albumin; 5, 125 mg of control peptide (sequenceETPEEREERRR) (SEQ ID NO:7). Panel B: D10 immunoprecipitations afterincubation with increasing amounts of g10 peptide. Lanes: 1, nocompetitor; 2, 37 nM; 3, 74 nM; 4, 148 nM; 5, 222 nM; 6, 444 nM. PanelC: immunoprecipitations of a g10 containing fusion protein (g10-U1-A)using various competitors. Lanes: 1, no competitor; 2, preimmune rabbitserum; 3, 7.4 nM g10 peptide; 4, 0.7 nM D10 RNA; 5, 0.7 nM unrelated RNA(cDNA encoding loop sequence: ACGTTCGTCG) (SEQ ID NO:8). Panel D:immunoprecipitation of g10-U1A fusion protein after incubation withincreasing amounts of D10 RNA. Lanes: 1, no competitor; 2, 0.0175 nM; 3,0.035 nM; 4, 0.35 nM; 5, 0.7 nM; 6, 1.05 nM.

[0016]FIG. 6 shows immunoprecipitation of RNAs tagged with the D10 RNAepitope. In Vitro transcribed [³²P] radiolabeled RNA wasimmunoprecipitated with anti-g10 serum or with an anti-U1 RNA serum(EW). Bound RNA was analyzed using a denaturing 6% acrylamide gel andautoradiography: Panel A: The D10 DNA construct (FIG. 2) was cloned intothe BamHI site of PGEM-3zf (+) to produce tagged vector RNA, andtranscripts with different 3′ termini were generated with SP6 RNApolymerase. The anti-g10 serum was used to precipitate the followingtranscripts (lanes): 1, U1 RNA (negative control); 2, D10 RNA; 3-5,increasing lengths of D10 tagged vector RNA; 6-10, supernatants fromlanes 1-5, respectively. Approximate nucleotide sizes are indicated byarrows. Panel B: U1 RNA was tagged with the D10 epitope by replacingloop III, sequence CAAAUGU (SEQ ID NO:9), with the sequence UGGUGGAGCA(SEQ ID NO:10) (construct U1-3Dx). Lanes: 1, total deproteinized HeLacell RNA; 2, total HeLa cell RNA mixed with exogenous NEU1 transcript(wild-type U1 RNA sequence plus extra 3′ nucleotides from the U1 gene);3, total HeLa cell RNA mixed with exogenous U1-3Dx, plus extra 3′nucleotides; 4-6, RNA mixtures from lanes 1-3 precipitated with ananti-U1 RNA serum (EW); 7-9, RNA mixtures from lanes 1-3 precipitatedwith the anti-g10 serum; 10-11, same as lane 9, but U1-3Dx was diluted1:3 and 1:9, respectively; 12-14, RNA mixtures from lanes 1-3, exceptthat total HeLa cell extract was used instead of HeLa cell RNA andprecipitated with anti-g10 serum; 15-17, RNA mixtures from lanes 1-3,precipitated with pre-immune serum. Only 0.5% of the total RNA mixtureswere loaded in lanes 1-3, and these lanes were exposed three timeslonger than the other lanes. U1-3Dx consistently produced a doublet asobserved in lanes 3, 6, 9-11, and 14. For unexplained reasons, a smallamount of endogenous U1 RNA was detected in the presence of total HeLacell extract (lanes 12-14).

DETAILED DESCRIPTION OF THE INVENTION

[0017] Amino acid sequences disclosed herein are presented in the aminoto carboxy direction, from left to right. The amino and carboxy groupsare not presented in the sequence.

[0018] Nucleotide sequences are presented herein by single strand only,in the 5′ to 3′ direction, from left to right.

[0019] The term “epitope,” as used herein, refers to a portion of amolecule which has a three-dimensional structure on an exposed surfaceto which an antibody can specifically bind, whether in the context ofsaid molecule or as a portion thereof.

[0020] The term “immunogen,” as used herein, refers to a compoundcapable of eliciting an immune response, whether or not that compound isintentionally used to induce an immune response.

[0021] The term “antigen binding protein,” as used herein, refers themembers of the immunoglobulin superfamily. Members of the immunoglobulinsuperfamily include, but are not limited to, major histocompatibilitycomplex molecules, cell adhesion molecules (including both neuronal celladhesion molecules and cellular cell adhesion molecules) virus receptorssuch as picornavirus receptors (e.g., poliovirus receptors, rhinovirusreceptors), growth factor receptors (e.g., interleukin receptors,lymphokine receptors), T cell receptors (e.g., alpha-beta class andgamma-delta class T cell receptors), and antibodies. Antibodies and Tcell receptors are currently preferred.

[0022] The term “antibodies” as used herein refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodiesmay be monoclonal or polyclonal and may be of any species of origin,including (for example) mouse, rat, rabbit, horse, or human, or may bechimeric antibodies.. See, e.g., M. Walker et al., Molec. Immunol. 26,403-11 (1989). Antibodies may be recombinant monoclonal antibodiesproduced according to the methods disclosed in Reading U.S. Pat. No.4,474,893, or Cabilly et al., U.S. Pat. No. 4,816,567. Antibodies mayalso be chemically constructed according to the method disclosed inSegel et al., U.S. Pat. No. 4,676,980. The term antibodies furtherincludes fragments which retain the specific binding characteristics ofthe antibody from which they are derived, with such fragments including,for example, Fab, F(ab′)₂, and Fv fragments, and the correspondingfragments obtained from antibodies other than IgG. Such fragments areproduced by known techniques. For example, monoclonal Fab fragments maybe produced in Escherichia coli by recombinant techniques known to thoseskilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989).

[0023] 1. Methods of Making Nucleic Acid Epitopes.

[0024] As noted above, the present invention provides a method ofgenerating a nucleic acid molecule which is immunologicallycross-reactive with a non-nucleic acid immunogen by combining an antigenbinding protein which binds said immunogen with a degenerate pool ofnucleic acid species (i.e., under conditions which permit the binding ofa nucleic acid species to the antigen binding protein) and thenrecovering a nucleic acid species bound by the antigen binding proteinfrom the degenerate pool. An embodiment of this method is schematicallyillustrated in FIG. 1, the steps of which are explained in detail below.

[0025] Initially, suitable anti-peptide antibodies (e.g., anti-g10antibodies) are obtained. For example, polyclonal antibodies used tocarry out the present invention may be produced by immunizing a suitableanimal (e.g., rabbit, goat, etc.) with a non-nucleic acid immunogenantigen for which a nucleic acid epitope is desired, collecting immuneserum from the animal, and removing the polyclonal antibodies from theimmune serum, in accordance with known procedures. Monoclonal antibodiesused to carry out the present invention may be produced in a hybridomacell line according to the technique of Kohler and Milstein, Nature 265,495-97 (1975). For example, a solution containing the appropriateantigen may be injected into a mouse and, after a sufficient time, themouse sacrificed and spleen cells obtained. The spleen cells are thenimmortalized by fusing them with myeloma cells or with lymphoma cells,typically in the presence of polyethylene glycol, to produce hybridomacells. The hybridoma cells are then grown in a suitable media and thesupernatant screened for monoclonal antibodies having the desiredspecificity. Variations and refinements of these techniques can beemployed to produce other types of antibodies, as noted above.

[0026] As an alternative to antibodies, other members of theimmunoglobulin superfamily such as T cell receptors may be employed, asnoted above. T cell receptors are structurally and functionallyanalogous to antibodies, and can be manipulated in much the same way asantibodies. See generally A. Williams and A. Barclay, Ann. Rev. Immunol.6, 381-405 (1988); S. Brostoff and M. Howell, Clin. Immunol. &Immunopathol. 62, 1-7 (1992).

[0027] Non-nucleic acid immunogens other than peptides which may beemployed include glycoproteins, fats, lipids, viruses (e.g.,rhinovirus), polysaccharides, carbohydrates, and allergens. Allergensinclude pollen, mold, spores, insects, epidermal particles, dust, etc.See, e.g., Greer Laboratories, Inc., Allergenic Extracts AllergySupplies & Services, 2-4 (Apr. 1, 1992) (Greer Laboratories, Inc., P.O.Box 800, Lenoir, N.C., USA 28645-0800; tel. 704-754-5327). Peptides arepreferred, with the term “peptide” as used herein referring to a peptideas a discrete molecule or residing in a protein.

[0028] As an alternative to immunizing an animal with a knownnon-nucleic acid immunogen, antibodies may be collected from a human oranimal subject without prior specific immunization to produce a nucleicacid epitope to an antigen binding protein where the native epitopebound by that antigen binding protein is unknown. For example,antibodies may be collected from human or animal subjects afflicted withautoimmune disease to produce a nucleic acid epitope whichimmunologically cross-reacts with the self peptide targeted byautoantibodies in the disorder. Examples of such autoimmune diseases inhuman subjects include, but are not limited to, systemic lupuserythematosus, myasthenia gravis, and rheumatoid arthritis.

[0029] Once suitable antigen binding proteins are obtained, they arethen combined with a degenerate pool of nucleic acid species. Suchdegenerate pools are known, and may be produced in accordance with knowntechniques. See, e.g., Blackwell et al., Science 250, 1104-1110 (1990);S. Deutscher and J. Keene, Proc. Natl. Acad. Sci. USA 85, 3299 (1988);Joyce et al., Nucleic Acids Res. 17, 711-722 (1989); Oliphant et al.,Methods Enzymol. 155, 568 (1987). The pool may be formed of DNAmolecules or RNA molecules, with pools of RNA molecules currentlypreferred. The nucleotide bases which form the pool may optionally bemodified by methylation, O-methylation, provision of base analogues withatypical hydrogen bonding patterns, etc. In general, degenerate pools ofnucleic acids comprise a plurality of distinct nucleic acid species inan aqueous solution. Typically, from 16 to 10¹⁰ distinct nucleic acidspecies are included in the pool, depending on the number of nucleotidesbeing randomized. The precise number is not critical, though it ispreferred that the number be sufficiently high to approach completerepresentation of all the possible members of the randomly representedset. Individual nucleic acid species within the pool will be 2, 3, 4, 5,or 6 nucleotides in length or more. There is no particular upper limiton the length of the nucleic acid species, with nucleic acids of 50,100, or 200 or more nucleotides being suitable. The nucleic acid speciesmay be linear or may possess some form of secondary structure, such as astem and loop structure. Each nucleic acid species in the pool includesa degenerate segment of nucleotides, typically of 2, 3, or 4 up to about25 or 100 nucleotides, in which each degenerate nucleotide position israndomly assigned both with respect to the other nucleotides in thatsegment of that species and with respect to nucleotides occupying thesame position in other species in the degenerate pool. Note that“random” as used herein does not mean perfectly random: it merely meanssufficiently random to provide a plurality of distinct species in thedegenerate pool from which a particular species may be retrieved.Finally, each species in the degenerate pool may include non-randomsegments, such as primer segments or replication origins foramplification of the pool, though these segments may ultimately beremoved from the final selected species as discussed below.

[0030] Combining the anti-peptide antigen binding protein with thedegenerate pool may be facilitated by immobilizing the antigen bindingprotein on a solid support and contacting the degenerate pool (i.e., theaqueous solution carrying the degenerate pool) to the solid support, allin accordance with known techniques.

[0031] Typically, and as illustrated in FIG. 1, the step of combiningthe degenerate pool with the antigen binding protein is followed by thestep of separating nucleic acid species bound to said solid support(e.g., by washing away any unbound nucleic acid species, then elutingnucleic acid species bound to the solid support); then producing a poolof complementary nucleic acids from said nucleic acid species separatedfrom said solid support (e.g., reverse transcribing a pool of cDNAs froma DNA or RNA pool), then amplifying the pool of complementary nucleicacids to produce a subset degenerate pool of nucleic acid species, andthen repeating the step of combining the degenerate pool of nucleic acidspecies with the antigen binding protein with the subset degenerate poolof nucleic acid species to produce a further subset degenerate pool ofnucleic acids. This sequence of steps may be cyclically repeated toproduce numerous subset degenerate pools, with the number of cyclestypically being from three to nine, though a single cycle may in manycases be sufficient.

[0032] A separating step as described above preferably includes a washstep and an elution step. The wash step removes undesired nucleic acidspecies from the solid support, and the elution step removes the desirednucleic acid species from the solid support to provide the subsetdegenerate pool. The elution step may be carried out by any suitablemeans, such as phenol extraction. The separating step may be carried outat the same wash stringency at each cycle (i.e., as either a highstringency or low stringency wash), or the wash stringency may bechanged between cycles (with stringency typically being adjusted fromlow stringency to high stringency as the cycles progress). In somecases, at least one high stringency wash step is included, and where theseparating step is repeated, a high stringency wash step is included asthe last separating step. Wash stringency may be increased by increasingthe concentration of NaCl or urea in the wash buffer or by increasingthe temperature of the wash buffer. Typically, buffers containing 150 mMNaCl at 4° C. are considered to provide lower wash stringency, buffersas above containing 0.3 M NaCl or greater, or 0.3 M urea or greater, orat temperatures greater than 20° C. are considered to provideintermediate to higher wash stringency, and buffers containing 0.5 MNaCl or greater, or 0.5 M urea or greater, or at temperatures greaterthan 37° C. are considered to provide higher wash stringency. Standardwashing buffers also contain 0.05% nonidet P-40 and 50 mM Tris-HCl at pH7.4, although the detergent, buffer, buffer salts, buffer concentration,and the pH are not critical and can be varied over a wide range. See,e.g., E. Harlow and D. Lane, Antibodies, A Laboratory Manual (ColdSpring Harbor Laboratory 1988): R. Bentley and J. Keene, Mol. Cell.Biol. 11, 1829 (1991).

[0033] The amplifying step may be carried out in vivo or in vitro by anysuitable means. See generally D. Kwoh and T. Kwoh, Am. Biotechnol. Lab.8, 14-25 (1990). In vivo amplification may be carried out by standardrecombinant DNA techniques, such as by ligating cDNA produced asdescribed above into a plasmid, and then taking that plasmid or poolthereof with inserts and transforming a bacterial culture therewith.See, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual(2d Ed. 1989) (Cold Spring Harbor Laboratory); J. Ma and M. Ptashne,Cell 51, 113-119 (1987); S. Deutscher and J. Keene, Proc. Natl. Acad.Sci. USA 85, 3299 (1988). Examples of suitable in vitro amplificationtechniques include, but are not limited to, polymerase chain reaction(see U.S. Pat. Nos. 4,683,202 and 4,683,195 to K. Mullis et al.), ligasechain reaction (see R. Weiss, Science 254, 1292 (1991)), stranddisplacement amplification (see G. Walker et al., Proc. Natl. Acad. Sci.USA 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res. 20,1691-1696 (1992)), transcription-based amplification, (see D. Kwoh etal., Proc. Natl. Acad Sci. USA 86, 1173-1177 (1989)), self-sustainedsequence replication (or “3SR”) (see J. Guatelli et al., Proc. Natl.Acad. Sci. USA 87, 1874-1878 (1990)), the Qβ replicase system (see P.Lizardi et al., BioTechnology 6, 1197-1202 (1988)), nucleic acidsequence-based amplification (or “NASBA”) (see R. Lewis, GeneticEngineering News 12 (9), 1 (1992)), the repair chain reaction (or “RCR”)(see R. Lewis, supra), and boomerang DNA amplification (or “BDA”) (seeR. Lewis, supra).

[0034] Once a desired nucleic acid species is recovered, it may beamplified and/or sequenced and synthesized in accordance with knowntechniques. A complementary nucleic acid (e.g., a cDNA) to the nucleicacid species may be produced by reverse transcription and the desirednucleic acid species produced in greater quantities by recombinanttechniques. The immunological cross-reactivity of the recovered nucleicacid species with the non-nucleic acid immunogen it mimics may beconfirmed by suitable immunoassay, such as blocking assays orcompetition experiments, carried out in accordance with knowntechniques.

[0035] The foregoing method provides an isolated nucleic acid whichinhibits complex formation between an antigen binding protein and anon-nucleic acid immunogen. Binding of the nucleic acid to such anantigen binding protein can be routinely determined in a standardcompetition assay in vitro, with nucleic acids of the invention havingdissociation constants (K_(d)s) of 10⁻⁵, 10⁻⁷ or 10⁻⁸ up to 10⁻¹² or10⁻¹⁴ moles per liter. The format of competition assay is not critical,though enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay(RIA) are particularly convenient. Nucleic acids of the invention haveassociation constants (K_(a)s) which make them useful as inhibitors ofthe binding of non-nucleic acid immunogens to antigen binding proteins(such as antibodies), with the K_(a)s for such nucleic acids rangingfrom 10⁵, 10⁷, 10⁸, or 10 ⁹ up to 10¹⁰ or 10¹² liters per mole, withthese values being determinable in the same manner as given above withrespect to K_(d)s. The nucleic acid itself may mimic any of a broadvariety of non-nucleic acid immunogens: for example, one embodiment ofthe foregoing is an isolated nucleic acid which inhibits complexformation between a self peptide autoantigen and an antigen bindingprotein, wherein said antigen binding protein is from a human or animalsubject which expresses said self peptide. Such antigen binding proteinsmay be obtained from human subjects afflicted with an autoimmunedisease, as noted above.

[0036] As with the nucleic acid species in the degenerate pool, isolatednucleic acids of the present invention may be of any length, typicallyof from 2, 3, 4, 5, or 6 nucleotides in length or more. Again there isno particular upper limit on the length of the isolated nucleic acid,with nucleic acids of 50, 100, or 200 or more nucleotides beingsuitable. As above, the isolated nucleic acid may be linear or maypossess some form of higher order structure, such as a stem and loopstructure. Further, the isolated nucleic acid may be modified from thatinitially retrieved from the degenerate pool, such as by removing primersegments or other portions thereof which are not critical for binding,or by making minor modifications to the structure of one or more of theindividual nucleotides in the nucleic acid itself such as methylation,O-methylation, provision of nucleotide analogues with atypical patternsof hydrogen bonding, other modifications to prevent nucleophilic attackon the phosphodiester bond, and the like.

[0037] 2. Uses for Nucleic Acid Epitopes.

[0038] Isolated nucleic acids of the invention can be used in a varietyof ways. For example, the isolated nucleic acid may be conjugated,either directly or indirectly and either covalently or non-covalently,to a molecule to be tagged thereby (i.e., a “tagged molecule”). Thetagged molecule itself may be, for example, a protein or a heterologousnucleic acid. The tagged molecule can then be detected with antigenbinding proteins, particularly antibodies, known to bind that isolatednucleic acid.

[0039] Nucleic acids of the invention may be used in methods ofdetecting an antigen binding protein which binds a predeterminednon-nucleic acid immunogen. Such methods comprise contacting abiological sample suspected of containing the antigen binding protein toa nucleic acid, which nucleic acid is capable of inhibiting complexformation between the antigen binding protein and said non-nucleic acidimmunogen, under conditions which permit the formation of a reactionproduct; and then detecting the presence or absence of the reactionproduct. Biological samples taken from human or animal subjects for usein this method are generally biological fluids such as serum, bloodplasma, or ascites fluid. In the alternative, the sample taken from thesubject can be a tissue sample (e.g., biopsy tissue; scrapings; etc.).Any suitable assay format can be used to carry out the detection of thereaction product, examples being radioimmunoassay, immunofluorescencemethods, enzyme-linked immunoassays, and the like. Those skilled in theart will be familiar with numerous specific immunoassay formats andvariations thereof which may be useful for carrying out the methoddisclosed herein. See generally E. Maggio, Enzyme-Immunoassay,(1980)(CRC Press, Inc., Boca Raton, Fla.).

[0040] Nucleic acids of the present invention may be used to produce animmune response to a non-nucleic acid immunogen in a human or animal(e.g., dog, cat, horse, goat, rabbit) subject. In this case, the nucleicacid serves as a surrogate immunogen for the non-nucleic acid immunogen.The method comprises administering a nucleic acid to the subject, whichnucleic acid is capable of inhibiting complex formation between anantigen binding protein and the immunogen, with the nucleic acid beingadministered in an amount effective to induce an immune response in saidanimal to said immunogen. Techniques for enhancing the immunogenicity ofthe nucleic acid which are known in the art may, if desired, beemployed. Subjects may be administered nucleic acids for this purpose tosimply raise stocks of antibodies, or for therapeutic purposes tosubjects in need of such treatment. Administration to a subject may becarried out by any suitable means, such as by subcutaneous injection,intravenous injection, intraperitoneal injection, and nasal spray. Theamount of nucleic acid administered will depend upon factors such asroute of administration, species, use of booster administrations, etc.,but is generally between 50 micrograms to 5 milligrams per kilogramsubject body weight, and more typically is between 50 micrograms and 200micrograms per kilogram subject body weight.

[0041] Nucleic acids of the invention may be employed in methods ofblocking complex formation between a non-nucleic acid immunogen and anantigen binding protein (again, typically an antibody) which binds thenon-nucleic acid immunogen. Such methods comprise contacting the antigenbinding protein to a nucleic acid, which nucleic acid inhibits complexformation between the antigen binding protein and the non-nucleic acidimmunogen. The contacting step may be carried out in vitro (againtypically by combining constituents in an aqueous solution), or may becarried out in vivo in a human or animal subject. Where carried out invivo, the subject, dosage, route of administration, and other parametersmay be as given above in connection with a method of inducing an immuneresponse. Where carried out in vitro, again, numerous different formatsfor carrying out such blocking experiments will be known to thoseskilled in the art, as also discussed above.

[0042] Nucleic acids may be prepared for administration to a subject asa pharmaceutical composition comprising the nucleic acid in apharmaceutically acceptable carrier. Preparation is typically carriedout by intimately admixing the nucleic acid with the carrier. Thenucleic acid is included in an amount sufficient to achieve the intendedeffect: i.e., induce an immune response or block complex formationbetween an antigen binding protein and an immunogen, as explained above.Pharmaceutically acceptable carriers may be solid or liquid carriers,such as sterile pyrogen-free phosphate-buffered saline solution. Thecarrier may optionally contain one or more adjuvants, such as aluminumhydroxide, aluminum phosphate, plant and animal oils, etc. In addition,the vaccine formulation may contain one or more stabilizer, exemplarybeing carbohydrates such as sorbitol, mannitol, starch, sucrose,dextran, and glucose, proteins such as albumin or casein, and bufferssuch as alkaline metal phosphates and the like.

[0043] 3. Generation of Nucleic Acid Sets Useful as Polyvalent Vaccines.

[0044] The techniques described above may be used to provide a method ofgenerating a set of nucleic acid species useful as immunogens, where atleast two members of the set are not immunologically cross-reactive withone another. By “immunologically cross-reactive” is meant that thecross-reactive compounds or species bind to the same antigen bindingprotein at the same site (including overlapping sites) with the bindingaffinities give herein (e.g., they compete for binding with oneanother). The method comprises collecting a plurality of antigen bindingproteins from a human or animal subject, which antigen binding proteinsare either antibodies or T cell receptors; combining the antigen bindingproteins with a degenerate pool of nucleic acid species; and thenrecovering a plurality of nucleic acid species bound by the antigenbinding proteins from the degenerate pool to produce the set.Preferably, the nucleic acid species are immunologically cross-reactivewith compounds which are not nucleic acids, and at least two members ofthe set are immunologically cross-reactive with different immunogens.Such methods are particularly useful for developing polyvalent vaccines,i.e., vaccines capable of inducing an immune response to differentepitopes on a common immunogen and/or vaccines capable of inducing animmune response to different immunogens.

[0045] The collecting step may be carried out by any suitable means,such as by harvesting T cell lymphocytes from the subject, by collectingimmune serum from the subject, or both. Note that the type of immuneresponse elicited by the vaccine can be biased towards cellular orhumoral by means of the type of antigen binding protein collected. If avaccine for a particular disease is desired the antigen binding proteinsmay be collected from a subject afflicted with the disease or byimmunizing a suitable subject with an immunogen for that disease priorto the collecting step. Note that this method provides the advantagethat an infectious agent or toxin need not be isolated or identified inorder to generate a vaccine against it. The technique may be employedwith any disease, including diseases of viral, bacterial, protozoan, orother microbial origin.

[0046] In an alternative embodiment, antigen binding proteins collectedfrom a plurality of subjects, all previously afflicted with the samedisease or infected with the same disease-causing organism, are pooledto ultimately provide a polyvalent vaccine directed against numerousvariants or serotypes of that disease. For example, one or more elderlysubjects who lived through the influenza epidepmic of 1918 will have thecombined immunological experience to resist any potential challenge bythe swine flu virus, H1N1. The combined immunological repertoire of thispopulation of individuals, which includes antibodies to multipledeterminants of the virus such as the surface antigens, hemagglutinin(H1) and neuraminidase (N1), can be captured from their pooled serausing this invention and that repertoire passed by immunization of animmunologically naive subject(s) using the polyvalent vaccine. Further,the entire immunological repertoire of any population which hasdeveloped immune resistance to any pathogen, known or unknown, could besimilarly passaged using such polyvalent vaccines derived from theircombined sera. It is one advantage of this invention that the vaccinerecipients need not be subject to exposure to the pathogen or to anymaterials extracted from a pathogen. The nucleic acid vaccine isrendered pure by simple extraction using any of several standardprocedures such as emulsification in phenol.

[0047] Another example of the practice of this invention is usingrhinoviruses which cause the common cold. It has not been possible todevelop satisfactory vaccines to rhinoviruses because approximately 30different, but related serotypes exist. The viral antigenic determinantscan change abruptly and mutate in order to escape immune survaillance.However, in the combined experience of a population of individualsresides a record of immunological response to all of the variousserotypes of the rhinovirus. Thus, the present invention will allow thederivation of a polyvalent vaccine to rhinovirus using the combined seraof a population of individuals experienced with infection by the manyserotypes of rhinovirus. Such a vaccine will provide broad protection tothe recipient for all rhinovirus challenges.

[0048] This invention embodies an additional advantage for theproduction of a polyvalent vaccine in that the selected nucleic acidmimetics will contain in the sub pool structural variants of theoriginal immunogen which can be used to immunize the subject againstunanticipated variants of a pathogen. For example, the protozoan agentof African sleeping sickness, Trypanosoma brucei, contains on itssurface VSG antigens that can undergo spontaneous antigen switchingallowing the pathogen to escape immune survellance. The presentinvention provides for the derivation of polyvalent nucleic acidvaccines which mimic subtle variations of the original antigen. Thus,some selected ligands in the vaccine resemble the exact originalnon-nucleic immunogen while others resemble subtle variations of theoriginal immunogen.

[0049] These methods provide a set of isolated nucleic acids, each ofwhich nucleic acids inhibits complex formation between an antigenbinding protein and an immunogen, wherein the antigen binding protein iseither an antibody or a T cell receptor, and wherein at least twomembers of the set do not bind to the same antigen binding protein.Members of the set will have the characteristics as given above: e.g.,bind to the antigen binding protein at a K_(d) of from 10⁻⁵ or 10⁻⁷ to10⁻¹⁴ moles per liter. The set may be provided in an aqueous carriersolution, may be provided in the form of a cDNA library encoding the setas described above, or may be provided in a pharmaceutically acceptablecarrier as described above. The set is essentially free of other nucleicacids which do not possess such binding characteristics, though otheringredients can of course be added to the set which do not detract fromthe function thereof.

[0050] 4. Mimetic Conformational Selection for Rational Drug Design.

[0051] The techniques described above can also be adapted for generatingtools for the rational design of drug compounds. Such techniques areparticularly useful where other structural information on the drugcompound is unavailable. In general, the method generates a plurality ofnucleic acid species which are immunologically cross-reactive with adrug compound (which compound is not a nucleic acid, and which compoundpossesses at least two epitopes). The method comprises immunizing ananimal with the drug compound according to methods described above, thencollecting antigen binding proteins that bind the compound, thencombining the antigen binding proteins with a degenerate pool of nucleicacid species, and then recovering a plurality of nucleic acid moleculesbound by the antigen binding protein from the degenerate pool, whereinat least two of the nucleic acid species do not bind to the same antigenbinding protein. The method may be employed with any drug compound whichpresents a plurality of epitopes thereon, including (but not limited to)peptides, glycoproteins, fats, lipids, polysaccharides, andcarbohydrates, including chemical analogues thereof.

[0052] The foregoing techniques provide a set of isolated nucleic acidspecies which inhibits complex formation between an antigen bindingprotein and a drug compound as described above, wherein at least two ofthe nucleic acid species do not bind to the same antigen bindingprotein. The characteristics of the members of the set are as givenabove: i.e., they bind to the antigen binding protein at a K_(d) of from10⁻⁵ or 10⁻⁷ to 10⁻¹⁴ moles per liter. The set may be provided in anaqueous carrier solution, as a cDNA library encoding the same, or in apharmaceutical carrier. The set may be screened itself for drug analogs,or may be used to vaccinate a suitable host subject as discussed aboveto generate additional complementary mimetic surface ligands. Again, theset is essentially free of other nucleic acids which do not possess suchbinding characteristics, though other ingredients can of course be addedto the set which do not detract from the function thereof.

[0053] Mimetic conformational selection can be used to improve thebiological efficacy of a compound such as a receptor binding molecule,by providing a pool of structural variants which themselves possessbiological activity as agonists or antagonists. For example, insulinwhich binds to an insulin receptor can serve as the immunogen andinsulin-binding antibodies can be collected for use in conformationalselection. Mimetic nucleic acid ligands selected using the antibodiesare structural analogus of insulin and can also be utilized asfunctional analogs of insulin in biological assays and therapeuticregimens. Furthermore, structural analysis of selected mimetic ligandsby any of several known methods (i.e., co-crystallographic analysis)will provide a means to correlate variations in biological function ofthe mimetic molecule with its structural features. For example, mimeticnucleic acid ligands that always display receptor agonist activityshould conserve certain structural features. These surfaces can bemodeled against the original drug compound in order to rationallyengineer optimal drug design.

[0054] The foregoing is explained in greater detail in the followingnon-limiting examples.

EXAMPLE 1 Preparation and Characterization of an Antiserum Reactive withthe g10 Peptide

[0055] A thirteen amino acid peptide was synthesized representing theamino-terminus of g10-fusion proteins expressed from the Studier T7expression vectors (F. Studier, et al., Meth. Enzym. 185, 60-89 (1991))(peptide sequence: MASMTGGQQMGRC-carboxyl amide (SEQ ID NO:11),purchased from Multiple Peptide Systems). The first eleven amino acidsrepresent the gene 10 protein, the arginine is encoded by the linker inthese expression vectors, and the cysteine was incorporated forconjugation to the carrier. The peptide was coupled to keyhole limpethemocyanin (Sigma) using the crosslinker MBS(3-maleimidobenzoyl-N-hydroxysuccinimide ester, Boehringer MannheimBiochemicals). A high-titer antiserum was obtained from rabbitsimmunized with the peptide-carrier conjugate in accordance with standardtechniques. The specificity of the antiserum was characterized usingWestern blot and immunoprecipitation methods previously reported (J.Chambers and J. Keene, Proc. Natl. Acad. Sci. USA 82, 2115-2119 (1985);R. Bentley and J. Keene, Mol. Cell. Biol. 11, 1829-1839 (1991)), withdetails as set forth below. Recombinant proteins were expressed inbacteria using the Studier T7 system as published previously (supra).Western blots were probed with various sera diluted 1:2000 and decoratedwith [¹²⁵I] protein A. [³⁵S]-labeled proteins were prepared by in vitrotranscription of cDNA constructs with T7 RNA polymerase followed bytranslation in rabbit reticulocyte lysates.

[0056]FIG. 3 demonstrates that the serum was specific for proteinscontaining the g10 fusion peptide as assayed by immunoblot andimmunoprecipitation. FIG. 3A shows recognition by the anti-g10 serum ofg10-tagged U1-snRNP A protein (g10-U1-A) in Western blot analysis. Theanti-g10 serum reacted with only the g10-U1-A fusion protein (FIG. 3A,lane 6), but not with recombinant U1-A (lane 7) or authentic HeLa cellU1-A (lane 8), while a control anti-U1-A serum reacted withover-expressed U1-A and g10-U1-A, as well as with authentic HeLa cellU1-A (compare FIG. 3A, lanes 6-8 with 10-12). Anti-g10 reactivity withg10-U1-A protein was analyzed also by immunoprecipitation. As expected,the anti-g10 serum precipitated g10-U1-A fusion protein (FIG. 3B, lane7), but not U1-A protein (lane 6). Anti-g10 antibody binding could beinhibited by excess free g10 peptide (FIG. 3B, lanes 8-11), but not by acontrol peptide (lane 12), further demonstrating that the interactionwas specific for the g10 peptide sequence. Taken together, these resultsdemonstrate that the g10 fusion peptide can function as an epitope tagrecognized by the anti-g10 serum.

[0057] A more demanding test of an epitope tag is recognition of thetagged molecule within the context of a macromolecular complex. Amongthe potential complications of using an epitope tag are its interferencein assembly of a complex or its inaccessibility within an assembledcomplex. Previous studies have used the g10 tag to immunoprecipitate RNPcomplexes formed in vitro between g10-U1-A and U1 RNA. Other RNPcomplexes analyzed using the g10 epitope include those formed by U1-70K,U2-B″, U2-A′, and Ro-RNP 60 kD. RNP particles formed by these taggedproteins in vivo also are accessible to recognition of the epitope bythe anti-g10 serum as assessed by immunoprecipitation andimmunofluorescence (data not shown).

EXAMPLE 2 Selection of Specifically Bound RNA by Immunoprecipitationwith g10 Peptide Antibody

[0058] Whereas the g10 peptide is a useful epitope tag for analyzingcomplexes containing protein, an RNA epitope tag would be equally usefulfor studying complexes containing RNA. The anti-g10 serum was presentedwith a degenerate pool of RNA containing 1,048,576 unique species,assuming incorporation of four different nucleotides at 10 randomizedloop positions (FIG. 2). These RNAs were transcribed from approximately1×10¹¹ molecules of degenerate oligodeoxynucleotide template (D. Tsai etal., Nucl. Acids Res. 19, 4931-4936 (1991)). Assuming that mosttemplates are transcribed at least once, all possible RNA species shouldbe redundantly represented.

[0059] RNA was prepared by in vitro transcription of PCR-generatedtemplates in accordance with known techniques (D. Tsai et al., Nucl.Acids Res. 19, 4931-4936 (1991)). The transcripts wereimmunoprecipitated with the anti-g10 serum as follows: Protein ASepharose beads (Sigma, 4 mg per 50 ml reaction) were washed in NT2buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.05% Nonidet P-40) (C. Queryet al., Cell 57, 89-101 (1989)), mixed with 2 ml of anti-g10 serum,incubated on ice for 10 min, and washed in NT2. The beads were thenresuspended in 100 ml KNET+ buffer (50 mM Tris pH 7.4, 80 mM NaCl, 20 mMKCl, 2 mM EGTA, 0.05% Nonidet P-40, 1 mM MgCl₂, 2.5% polyvinyl alcohol,40 units/ml of RNasin, 5 mg/ml of poly(A) RNA, and 0.2% VRC, 50 mg/mltRNA and 50 mg/ml BSA) (C. Query et al., supra). RNA was added and thereactions incubated at 37° C. for 7 minutes. The pellets were washedfive times with NT2 buffer. Higher stringency washes included 0.5 M ureain the first wash. RNA was recovered by phenol extraction and ethanolprecipitation. RNAs were reverse transcribed and cDNAs subjected to PCRamplification as described previously (D. Tsai et al., Nucl. Acids Res.19, 4931-4936 (1991)). The amplified template was used to repeat theabove cycle for two additional rounds. The final PCR product wasdigested with BamHI, cloned and sequenced.

[0060] Low stringency selection yielded five different but relatedspecies (data not shown). On the other hand, after three cycles ofantibody selection with high stringency washing, a single RNA species,D10, was obtained in 45 out of 45 isolates.

[0061] To rule out the possibility of nonspecific RNA binding, [³²P]-D10RNA precipitations were performed with either protein A Sepharose beadsalone, pre-immune serum, or anti-g10 serum. The D10 RNA bound only tothe anti-g10 serum (FIG. 4, lane 1), confirming that binding is specificfor the post-immune antiserum. Immunoprecipitation experiments withvarious RNA species suggest that the anti-g10 serum is specific for theD10 RNA sequence. For example, an unrelated RNA was not recognized (FIG.4, lane 6), nor were RNAs containing the same stem but with differentloop sequences (lanes 5 and 7). These findings demonstrate that theantibody binds a specific RNA structure or sequence.

EXAMPLE 3 D10 RNA Binds to the g10 Antibody Antigen Recognition Site

[0062] Although the D10 RNA is recognized by the anti-g10 serum, theselection procedure theoretically could recover RNA bound to any surfaceof any antibody molecule in the serum. Since the pre-immune serum showedno reactivity towards the D10 RNA, the most likely RNA-binding surfaceis the antigen-combining site of the g10-specific antibodies. Thispossibility was tested by competition experiments using the g10 peptideand the D10 RNA.

[0063] Competition experiments were performed by addition of the variouscompetitors to [³²P ] labeled RNA or [35S] labeled in vitro translatedg10-snRNP-A fusion protein prior to immunoprecipitation. Bound RNAs wereanalyzed on 6% denaturing acrylamide gels and proteins on 10% SDS-PAGEgels, followed by autoradiography. Antibody-RNA complexes were formed inthe presence of competitor g10 peptide or control peptide (FIG. 5A), orwith increasing amounts of g10 peptide (FIG. 5B), and uncompeted RNA wasrecovered by immunoprecipitation. As expected, neither bovine serumalbumin nor an unrelated peptide showed any effect on D10 RNAprecipitation by the anti-g10 serum (FIG. 5A, lanes 4 and 5). However,increasing amounts of g10 peptide inhibited -RNA binding by theantiserum (FIG. 5B, lanes 1-6).

[0064] Similarly, the D10 RNA was examined for its ability to inhibitcomplex formation between a g10 fusion protein and the g10-reactiveantibodies. The D10 RNA was able to compete with the g10 fusion proteinfor binding to the antibody (FIG. 5C, lane 4). An unrelated RNA was notable to compete for the antibody combining site (FIG. 5C, lane 5). Inaddition, increasing amounts of D10 RNA were able to progressivelycompete with an [³⁵S] labeled g10 fusion protein (FIG. 5D, lanes 1-6).These results are consistent with competition between two antigens forthe same or overlapping antigen-binding sites on the antibody. Thefinding that essentially all of the reactive antibodies recognized boththe RNA and peptide epitopes implies that the immune response wasmounted against a single epitope on the g10 peptide and that the D10 RNAcontains a single cross-reactive epitope.

EXAMPLE 4 Use of the D10 RNA as an Epitope Tag

[0065] To test whether D10 can serve as an epitope tag for RNA, the D10DNA sequence (FIG. 2) was cloned into pGEM-3-zf(+) and various lengthtranscription templates produced by truncation 3′ to the D10 epitope.RNA was synthesized in vitro from these templates, and the anti-g10serum was used to immunoprecipitate the D10-tagged pGEM-3zf(+) RNAs. Allfusion RNAs were precipitated by the anti-g10 serum (FIG. 6A, lanes2-5), while a control RNA was not precipitated (lane 1). Therefore, theD10 sequence is functional as an RNA epitope tag in these contexts.

[0066] The complexity of RNA sequences within the pool ofartificially-randomized 10-mer loops approaches that of RNA sequenceswithin a HeLa cell; however, recognition of an RNA epitope within thecellular milieu may encounter different constraints than recognitionwithin a pool of in vitro transcripts. In an effort to assess theutility of the D10 epitope in cellular extracts, we immunoprecipitatedD10-tagged in vitro transcripts mixed with total HeLa cell RNA or withHeLa cell extracts.

[0067] [³²P] labeled HeLa cell extracts and [³²P] labeled deproteinizedHeLa cell RNA were prepared in accordance with known techniques (R.Bentley and J. Keene, Mol. Cell. Biol. 11, 1829-1839 (1991)). [³²P]labeled in vitro transcripts were mixed with either HeLa cell extract orHeLa cell RNA under the D10 binding conditions described above. Theanti-RNA reactivities in the anti-g10 serum and in the anti-U1 RNApatient serum (EW) were normalized by using 1 ml of a (1:5) dilution ofserum EW and 10 ml of the anti-g10 serum. Antibody-RNA complexes wereanalyzed as described above.

[0068] A construct of U1 RNA in which the loop III was replaced by theloop of D10 RNA, termed U1-3Dx (gift of J. Snedeker), was efficientlyprecipitated by the anti-g10 serum in the presence of total HeLa cellRNA (FIG. 6B, lanes 9-11) and extract (lane 14). In contrast,transcripts of exogenous NEU1 RNA (see legend to FIG. 6B) were notimmunoprecipitated by the anti-g10 serum (FIG. 6B, lane 8 and 13);however, they were recognized by a patient serum, EW, that binds thesecond stem-loop of U1 RNA (lane 5). Furthermore, the anti-g10 serum wasnot reactive with HeLa cell RNA (FIG. 6B, lane 7). These results showthat the D10 epitope can-be recognized in the presence of total cellularRNA and proteins. Furthermore, the fact that the U1-3Dx construct wasrecognized by the anti-g10 serum identifies a minimal sequence requiredfor antibody recognition as CCUGGUGGAGCAGG (SEQ ID NO:12), in thecontext of a stem.

EXAMPLE 5 Autoimmune Serum from an SLE Patient Binds RNA Sequences froma Degenerate Pool

[0069] This example demonstrates that antibodies taken directly from asubject afflicted with an autoimmune disease can be used to generatenucleic acid species which bind thereto.

[0070] A degenerate pool of RNA sequences is created using synthetic DNAoligomers that are randomized in either of three different contextsrepresenting linear unstructured RNA or in the framework of the naturalU1 stem II, from positions 50-89, containing degenerate loops of 10 or13 nucleotides as described in detail previously (D. Tsai et al., Nucl.Acids Res. 19, 4931-4936 (1991)). All three RNA structural contextsshare identical PCR primer regions at the 5′ and 3′ termini. A selectionprocedure is performed consisting of three cycles of successivetranscription, RNA immunoprecipitation with serum from a patientafflicted with systemic lupus erythematosus (SLE), reversetranscription, and PCR as described above. Sequencing of multipleclones, each representing a selected RNA species, reveals several RNAspecies which bind to antibodies in EW patient serum.

[0071] The foregoing examples are illustrative of the present invention,and are not to be construed as limiting thereof. The invention isdefined by the following claims, with equivalents of the claims to beincluded therein.

1 12 121 base pairs nucleic acid single linear cDNA 1 CGCGGATCCTAATACGACTC ACTATAGGGG CCACCAACGA CATTGGGCGA GGCTTATCCT 60 GGTGGAGCAGGATGTGCTGA CCCCGTTGAT ATAAATAGTG CCCATGGATC CGCGGGTGTC 120 G 121 83 basepairs nucleic acid single linear rRNA 2 GGGCCACCAA CGACAUUGGG CGAGGCUUAUCCNNNNNNNN NNGGAUGUGC UGACCCCGUU 60 GAUAUAAAUA GUGCCCAUGG AUC 83 83 basepairs nucleic acid single linear rRNA 3 GGGCCACCAA CGACAUUGGG CGAGGCUUAUCCUGGUGGAG CAGGAUGUGC UGACCCCGUU 60 GAUAUAAAUA GUGCCCAUGG AUC 83 12amino acids amino acid single linear peptide 4 Gly Lys Ser Arg Gly PheAla Phe Val Glu Phe Lys 1 5 10 10 base pairs nucleic acid single linearrRNA 5 CACCAUAUAA 10 10 base pairs nucleic acid single linear rRNA 6CUGACCCCGU 10 11 amino acids amino acid single linear peptide 7 Glu ThrPro Glu Glu Arg Glu Glu Arg Arg Arg 1 5 10 10 base pairs nucleic acidsingle linear cDNA 8 ACGTTCGTCG 10 7 base pairs nucleic acid singlelinear rRNA 9 CAAAUGU 7 10 base pairs nucleic acid single linear rRNA 10UGGUGGAGCA 10 13 amino acids amino acid single linear peptide 11 Met AlaSer Met Thr Gly Gly Gln Gln Met Gly Arg Cys 1 5 10 14 base pairs nucleicacid single linear rRNA 12 CCUGGUGGAG CAGG 14

That which is claimed is:
 1. A method of generating a nucleic acidspecies which is immunologically cross-reactive with an immunogen, whichimmunogen is not a nucleic acid, said method comprising: combining anantigen binding protein which binds said immunogen with a degeneratepool of nucleic acid species; and then recovering a nucleic acid speciesbound by said antigen binding protein from said degenerate pool.
 2. Amethod according to claim 1, wherein said antigen binding protein isselected from the group consisting of antibodies and T cell receptors.3. A method according to claim 1, wherein said antigen binding proteinis a monoclonal antibody.
 4. A method according to claim 1, wherein saidantigen binding protein is a polyclonal antibody.
 5. A method accordingto claim 1, wherein said antigen binding protein is an anti-g10antibody.
 6. A method according to claim 1, wherein said immunogen isselected from the group consisting of peptides, glycoproteins, fats,lipids, polysaccharides, carbohydrates, viruses and allergens.
 7. Amethod according to claim 1, wherein said immunogen is an allergen.
 8. Amethod according to claim 1, wherein said degenerate pool of nucleicacid species is a degenerate pool of RNA species.
 9. A method accordingto claim 1, wherein said degenerate pool of nucleic acid speciescomprises a plurality of nucleic acids having from 2 to 200 nucleotides.10. A method according to claim 1, wherein said degenerate pool ofnucleic acid species comprises a plurality of nucleic acids having from4 to 100 nucleotides.
 11. A method according to claim 1, wherein saiddegenerate pool of nucleic acid species comprises a plurality of nucleicacids having a degenerate segment of from 2 to 25 nucleotides.
 12. Amethod according to claim 1, wherein said degenerate pool of nucleicacid species comprises a plurality of nucleic acids in an aqueoussolution.
 13. A method according to claim 1, wherein said degeneratepool of nucleic acid species comprises a plurality of linear nucleicacids.
 14. A method according to claim 1, wherein said degenerate poolof nucleic acid species comprises a plurality of nucleic acids having astem and loop configuration.
 15. A method according to claim 1, whereinsaid nucleic acid species is an RNA species, said method furthercomprising the step of synthesizing a DNA encoding said RNA species fromsaid RNA species.
 16. A method according to claim 1, further comprisingthe step of assaying the immunological cross-reactivity of saidimmunogen and said nucleic acid species.
 17. A method according to claim1, further comprising the step of collecting said antibodies from ahuman or animal subject prior to said combining step.
 18. A methodaccording to claim 1, further comprising the step of producing saidantigen binding protein by immunizing an animal with said immunogen. 19.A method according to claim 1, further comprising the step of collectingsaid antibodies from a human or animal subject afflicted with anautoimmune disease.
 20. A method according to claim 1, furthercomprising the step of collecting said antibodies from a human subject,which human subject is afflicted with a condition selected from thegroup consisting of systemic lupus erythematosus, myasthenia gravis, andrheumatoid arthritis.
 21. A method according to claim 1, wherein saidantigen binding protein is immobilized on a solid support, and saidrecovering step is carried out by contacting said degenerate pool ofnucleic acid species to said solid support.
 22. A method according toclaim 21, wherein said contacting step is followed by the steps of:separating nucleic acid species bound to said solid support; thenproducing a pool of complementary nucleic acids from said nucleic acidspecies separated from said solid support; then amplifying said pool ofcomplementary nucleic acids to produce a subset degenerate pool ofnucleic acid species; and then repeating said step of contacting adegenerate pool of nucleic acid species to said solid support with saidsubset degenerate pool of nucleic acid species.
 23. A method accordingto claim 22, wherein said subset degenerate pool of nucleic acidsspecies bind to said antigen binding protein under conditionsrepresented by a wash stringency of 0.15M NaCl.
 24. A method accordingto claim 22, wherein said amplifying step is carried out in vivo.
 25. Amethod according to claim 22, wherein said amplifying step is carried ouin vitro.
 26. An isolated nucleic acid which inhibits complex formationbetween an antigen binding protein and an immunogen, which immunogen isnot a nucleic acid.
 27. An isolated nucleic acid according to claim 26,which immunogen is a peptide.
 28. An isolated nucleic acid according toclaim 26 which inhibits complex formation between a self peptideautoantigen and an antigen binding protein, wherein said antigen bindingprotein is from a human or animal subject which expresses said selfpeptide.
 29. An isolated nucleic acid according to claim 26, whichantigen binding protein is selected from the group consisting ofantibodies and T cell receptors.
 30. An isolated nucleic acid accordingto claim 26 which binds to said antigen binding protein at a K_(d) offrom 10⁻⁵ to 10⁻¹⁴ moles per liter.
 31. An isolated nucleic acidaccording to claim 26 consisting of from 2 to 50 nucleotides.
 32. Anisolated nucleic acid according to claim 26 which is linear.
 33. Anisolated nucleic acid according to claim 26 which has a stem and loopconfiguration.
 34. An isolated nucleic acid according to claim 26 whichbinds to an antibody which binds to the g10 protein.
 35. An isolatednucleic acid according to claim 26 conjugated to a tagged molecule, saidtagged molecule selected from the group consisting of proteins andheterologous nucleic acids.
 36. A method of detecting an antigen bindingprotein which binds a predetermined immunogen, which predeterminedimmunogen is not a nucleic acid, said method comprising: contacting abiological sample suspected of containing said antigen binding proteinto a nucleic acid, which nucleic acid is capable of inhibiting complexformation between said antigen binding protein and said immunogen, underconditions which permit the formation of a reaction product; and thendetecting the presence or absence of a reaction product.
 37. A methodaccording to claim 36, which antigen binding protein is an antibody. 38.A method according to claim 36, which immunogen is a peptide.
 39. Amethod according to claim 36, which nucleic acid consists of from 2 to50 nucleotides.
 40. A method of blocking complex formation between animmunogen and an antigen binding protein which binds said immunogen,wherein said immunogen is not a nucleic acid, said method comprising:contacting said antigen binding protein to a nucleic acid, which nucleicacid inhibits complex formation between said antigen binding protein andsaid immunogen.
 41. A method according to claim 40, which antigenbinding protein is selected from the group consisting of antibodies andT cell receptors.
 42. A method according to claim 40, which immunogen isa peptide.
 43. A method according to claim 40, which nucleic acidconsists of from 2 to 50 nucleotides.
 44. A method of producing animmune response to an immunogen in a human or animal subject, whichimmunogen is not a nucleic acid, said method comprising: administering anucleic acid to said subject, which nucleic acid is capable ofinhibiting complex formation between an antigen binding protein and saidimmunogen; said nucleic acid being administered in an amount effectiveto induce an immune respone in said animal to said immunogen.
 45. Amethod according to claim 44, which antigen binding protein is selectedfrom the group consisting of antibodies and T cell receptors.
 46. Amethod according to claim 44, which immunogen is a peptide.
 47. A methodaccording to claim 44, which nucleic acid consists of from 2 to 50nucleotides.
 48. A method according to claim 44, which nucleic acid isadministered in an amount ranging from 50 micrograms to 5 milligrams perkilogram subject body weight.
 49. A method of generating a set ofnucleic acid species useful as immunogens, and wherein at least twomembers of said set are not immunologically cross-reactive with oneanother, said method comprising: collecting a plurality of antigenbinding proteins from a human or animal subject, which antigen bindingproteins are selected from the group consisting of antibodies and T cellreceptors; combining said antigen binding proteins with a degeneratepool of nucleic acid species; and then recovering a plurality of nucleicacid species bound by said antigen binding proteins from said degeneratepool to produce said set.
 50. A method according to claim 49, wherein aplurality of said nucleic acid species are immunologicallycross-reactive with compounds which are not nucleic acids.
 51. Amethod-according to claim 49, wherein at least two members of said setare immunologically cross-reactive with different immunogens.
 52. Amethod according to claim 49, wherein at least two members of said setare immunologically cross-reactive with a different immunogen, each ofwhich different immunogen is not a nucleic acid.
 53. A method accordingto claim 49, wherein said collecting step is carried out by harvesting Tcell lymphocytes from said subject.
 54. A method according to claim 49,wherein said collecting step is carried out by collecting immune serumfrom said subject.
 55. A method according to claim 49, furthercomprising the step of producing said antigen binding proteins byimmunizing said subject with an immunogen prior to said collecting step.56. A method according to claim 49, wherein said subject has beenafflicted with a microbial disease.
 57. A method according to claim 49,wherein said subject has been afflicted with a viral disease.
 58. Amethod according to claim 49, wherein said antigen binding proteins areimmobilized on a solid support, and said recovering step is carried outby contacting said degenerate pool of nucleic acid species to said solidsupport.
 59. A method according to claim 58, wherein said contactingstep is followed by the steps of: separating nucleic acid species boundto said solid support; then producing a pool of complementary nucleicacids from said nucleic acid species separated from said solid support;then amplifying said pool of complementary nucleic acids to produce asubset degenerate pool of nucleic acid species which subset degeneratepool comprises said plurality of nucleic acid species; and thenrepeating said step of contacting a degenerate pool of nucleic acidspecies to said solid support with said subset degenerate pool ofnucleic acid species.
 60. A method according to claim 59, wherein saidsubset degenerate pool of nucleic acid species bind to said antigenbinding proteins under conditions represented by a wash stringency of0.15M NaCl.
 61. A set consisting essentially of isolated nucleic acids,each of which nucleic acids inhibits complex formation between anantigen binding protein and an immunogen, wherein said antigen bindingproteins are selected from the group consisting of antibodies and T cellreceptors, and wherein at least two members of said set do not bind tothe same antigen binding protein.
 62. A set according to claim 61,wherein at least two members of said set inhibit complex formationbetween an antigen binding protein and an immunogen, which immunogen isnot a nucleic acid.
 63. A set according to claim 61, wherein at leasttwo members of said set inhibit complex formation between an antigenbinding protein and an immunogen, which immunogen is not a nucleic acid,and wherein said at least two members bind to antigen binding proteinswhich do not bind to the same immunogen.
 64. A set according to claim61, wherein; at least two members of said set are immunologicallycross-reactive with a different immunogen, each of which immunogens isnot a nucleic acid.
 65. A set of isolated nucleic acid species accordingto claim 61, wherein said each of said nucleic acid species bind to saidantigen binding protein at a K_(d) of from 10⁻⁵ to 10⁻¹⁴ moles perliter.
 66. A set of isolated nucleic acid species according to claim 61in an aqueous carrier solution.
 67. A cDNA library encoding a set ofisolated nucleic acid species according to claim
 61. 68. A method ofgenerating a plurality of nucleic acid species which are immunologicallycross-reactive with a drug compound and are useful for rational drugdesign, which compound is not a nucleic acid, said method comprising:combining an antigen binding protein which binds said compound with adegenerate pool of nucleic acid species; and then recovering a pluralityof nucleic acid molecules bound by said antigen binding protein fromsaid degenerate pool and wherein at least two of said nucleic acidspecies do not bind to the same antigen binding protein.
 69. A methodaccording to claim 68, wherein said antigen binding protein is selectedfrom the group consisting of antibodies and T cell receptors.
 70. Amethod according to claim 68, wherein said compound is selected from thegroup consisting of peptides, glycoproteins, fats, lipids,polysaccharides, and carbohydrates.
 71. A method according to claim 68,wherein said antigen binding protein is immobilized on a solid support,and said recovering step is carried out by contacting said degeneratepool of nucleic acid species to said solid support.
 72. A methodaccording to claim 68, wherein said contacting step is followed by thesteps of: separating nucleic acid species bound to said solid support;then producing a pool of complementary nucleic acids from said nucleicacid species separated from said solid support; then amplifying saidpool of complementary nucleic acids to produce a subset degenerate poolof nucleic acid species, which subset degenerate pool comprises saidplurality of nucleic acid species; and then repeating said step ofcontacting a degenerate pool of nucleic acid species to said solidsupport with said subset degenerate pool of nucleic acid species.
 73. Aset consisting essentially of isolated nucleic acid species whichinhibit complex formation between an antigen binding protein and a drugcompound, which compound is not a nucleic acid, and wherein at least twoof said nucleic acid species do not bind to the same antigen bindingprotein.
 74. A set of isolated nucleic acid species according to claim73, which compound is selected from the group consisting of peptides,glycoproteins, fats, lipids, polysaccharides, and carbohydrates.
 75. Aset of isolated nucleic acid species according to claim 73 whichisolated nucleic acid species bind to said antigen binding protein at aK_(d) of from 10⁻⁵ to 10⁻¹⁴ moles per liter.
 76. A set of isolatednucleic acid species according to claim 73 in an aqueous carriersolution.
 77. A cDNA library encoding a set of isolated nucleic acidspecies according to claim 73.